Multiplex screening for lysosomal storage disorders (LSDS)

ABSTRACT

A novel protein profiling method of testing for Lysosomal Storage Diseases (“LSD”) using discovered normalized lysosomal fingerprint patterns. The fingerprint patterns reveal the health of lysosomal organelles, specific LSD, and clinical severity. Multiplexing bead technology for simultaneous screening of multiple LSD and normalizing measured enzyme activity or protein levels against other lysosomal proteins, enzymes, or enzyme activities. Compounds, reagents, and methods for identifying and quantifying multiple target enzymes and proteins.

RELATED APPLICATIONS

This application is a divisional application of, and claims priority to,U.S. patent application Ser. No. 10/551,396, now U.S. Pat. No.8,173,443, entitled “Multiplex Screening for Lysosomal StorageDisorders,” filed on Oct. 19, 2006, which is a 371 application based onPCT Application No. PCT/AU04/00403 filed Mar. 31, 2004, which claimspriority to the following applications: (1) Australian ProvisionalPatent Application Serial Number 2003/901451, entitled “AN IMPROVEDMETHOD OF SCREENING FOR LYSOSOMAL STORAGE DISORDERS,” filed on Mar. 31,2003, having Hopwood et al., listed as inventors; (2) AustralianProvisional Patent Application, Serial Number 2003/904174, entitled“MULTIPLEX SCREENING FOR LSD'S,” filed on Aug. 8, 2003, having Hopwoodet al., listed as inventors; (3) Australian Provisional PatentApplication, Serial Number 2003/904720, entitled “MULTIPLEX SCREENINGFOR LSD'S,” filed on Sep. 2, 2003, having Hopwood et al., listed asinventors. The entire content of each of the above identifiedapplications is hereby incorporated by reference.

BACKGROUND

The present invention is generally related to diagnostics that determineLysosomal Storage Disorders (“LSDs”) and related diseases in a subject.More particularly, this invention pertains to compounds, reagents, andmethods for identifying and quantifying the levels and ratios ofmultiple target antigens that are used to accurately diagnose LSD. Thetarget antigens are naturally present in biological fluids or tissues ofeither LSD or non-LSD patients.

LSDs represent a group of over 40 distinct genetic diseases thatgenerally affect young children. Individuals that are affected with aTSD present a wide range of clinical symptoms that depend upon thespecific disorder or a particular genotype involved. The clinicalsymptoms associated with LSD's can have a devastating impact on both thechild and the family of affected individuals. For example, centralnervous system dysfunction, behavioral problems, and severe mentalretardation are characteristic of many LSDs. Other clinical symptoms mayinclude skeletal abnormalities, organomegaly, corneal clouding anddysmorphic features (Neufeld and Muenzer, 1995). Patients are usuallyborn without the visible features of a LSD, but early stage symptoms canquickly develop into a progressive clinical concern. In severe cases,the affected children require constant medical management but stilloften die before adolescence.

The significance of LSDs to health care becomes obvious when comparingthe group incidence rate for a LSD (1:5,000 births) to the groupincidence rate of other with well-known and intensively studied geneticdisorders, such as phenylketonuria (1:14,000) and cystic fibrosis(1:2,500), wherein these figures reflect incidence rates for Caucasianpopulations.

Once an individual begins to present the symptoms of a LSD, the actualclinical diagnosis of the disease is still a complex process. A clinicaldiagnosis of a LSD often requires multiple visits to a range ofspecialists, which can take months or even years. This long process isextremely stressful on the patient and family. Fortunately, there hasbeen considerable progress in the diagnosis of LSDs over the past 20years. For example, the development and introduction ofchromatographic-based urine screens for a specific group of LSDs calledmucopolysaccharidoses (“MPS”) and oligosaccharidoses has facilitatedscreening of clinically selected patients for these disorders. Followinga clinical index of suspicion for the disorders, the next stage ofdiagnosis involves a urine screen, wherein a “positive” urine screen isthen followed by specific enzymatic analysis. Although thechromatographic-based screening methods are simple to perform, they arerelatively labor-intensive and often require experience to accuratelyinterpret results. One example includes a method of identifying andquantitating biochemical markers (“biomarkers”) that are present inbiological fluids or tissues of a patient having a MPS or relateddisorders comprises determining a target quantity of a target MPSbiomarker oligosaccharide from a target biological sample taken from thetarget animal, and then comparing the target quantity to a referencequantity of a reference MPS biomarker oligosaccharide for the diagnosis,characterization, monitoring, and clinical management of MPS and relateddisease, as described in PCT Application AU03/00731 entitled“identification of Oligosaccharides and their Use in the Diagnosis andEvaluation of Mucopolysaccharidoses and Other Related Disorders,” filedon Jun. 13, 2003 with Hopwood et al., listed as inventors (the entirecontent of PCT Application AU03/00731 is hereby incorporated byreference). Consequently, chromatographic-based screening tests for LSDsare not used in some centers. Furthermore, these chromatographic-basedscreens are not readily amenable to automation, which has furtherlimited their utilization in screening strategies for newborns.

The production of specific substrates and antibody capture assays hasmade the enzymatic analyses for LSDs more accurate. Although not wantingto be bound by theory, the majority of LSDs result from a reduction inlevels of a particular enzyme(s) involved in a specific LSD, and theidentification of the specific enzyme(s) steady state in normalindividuals will help identify the particular form of LSD in theaffected individual. The ability to quickly and accurately determine thelevels of the more than 40 enzymes known to be involved with LSDs willassist in the development of better and more economical screeningassays. Unfortunately, many of the chromatographic-based screens andenzyme assays mentioned above are time-consuming, invasive, complex, andrequire cultured cells, or tissue biopsies, which tends to make suchassays inconvenient and expensive. As a result, testing for a LSD isoften not a first line strategy for an affected child with early stagesymptoms. Newborn screening for LSDs promises to provide early detectionof the LSD, but all newborns must be screened in order to detect thedisease early. Patients having a family history of LSDs may have ajustifiable reason to perform an early screen for a LSD. However, thecost of an early screen of the LSD in individuals not having a familyhistory may not be justified economically. Therefore, it would bebeneficial that any LSD screening process be capable of economicallyscreening large numbers of newborns.

One common feature of LSDs is the accumulation and storage of materialswithin lysosomes. It is generally recognized that the accumulation andstorage of material in LSD affected individuals results in an increasein the number and the size of lysosomes within a cell from approximately1% to as much as 50% of total cellular volume. In non-affectedindividuals, such materials are typically degraded into degradationproducts within the lysosome and then transported across the lysosomalmembrane. Certain lysosomal proteins are present at elevated levels inthe lysosomes of affected individuals (Meikle et al., 1997; Hua et al.,1998). These identified proteins are useful biomarkers for an earlydiagnosis of all LSDs. For example, sensitive immunoquantificationassays have been developed to monitor the level of useful biomarkerssuch as the lysosome-associated membrane proteins (“LAMPs”), saposins,and α-glucosidase. Although the determination of either LAMP-1 or LAMP-2levels alone in an ‘at-increased-risk’ group will identify up to 65% ofLSD affected individuals, the combination of a LAMP with one of thesaposins increase identification of LSD affected individuals toapproximately 85%. Therefore, a method to identify two or morebiomarkers simultaneously would increase the accuracy of diagnosing aspecific LSD as compared to any single assay. An automated multiplexassay that could perform a simultaneous screen on each of the known LSDdeficient enzymes would reduce time and cost for accurate LSD diagnosis.

Multiplexing Bead Technology is built around 3 core technologies. Thefirst is the family of fluorescently dyed microspheres having specificbiomolecules bound to the surface of the microsphere. The second is aflow cytometer with 2 lasers and associated optics to measurebiochemical reactions that occur on the surface of the microspheres, andthe third is a high-speed digital signal processor to efficiently managethe fluorescent output. This type of system has been described in, forexample: U.S. Pat. Nos. 6,449,562; 6,524,793 and U.S. patent applicationSer. No. 09/956,857. U.S. Pat. No. 6,449,562 (“the '562 patent”)entitled “Multiplexed Analysis of Clinical Specimens Apparatus, andMethod,” having Chandler et al. listed as inventors was issued on Sep.10, 2002. The '562 patent discloses a method for the multiplexeddiagnostic and genetic analysis of enzymes, DNA fragments, antibodies,and other biomolecules comprising the steps of constructing anappropriately labeled headset, exposing the headset to a clinicalsample, and analyzing the combined sample/beadset by flow cytometry.Flow cytometric measurements are used to classify, in real-time, beadswithin an exposed headset and textual explanations, based on theaccumulated data obtained during real-time analysis, are generated forthe user. The inventive technology of the '562 patent enables thesimultaneous, and automated, detection and interpretation of multiplebiomolecules or DNA sequences in real-time while also reducing the costof performing diagnostic and genetic assays. However, the '562 patentdoes not describe how to utilize the technology for diagnosing LSD's.

U.S. Pat. No. 6,524,793 (“the '793 patent”) entitled “MultiplexedAnalysis of Clinical Specimens Apparatus and Method,” having Chandler etal. listed as inventors, was issued on Feb. 25, 2003. The '793 patentdiscloses a method for the multiplexed diagnostic and genetic analysisof enzymes, DNA fragments, antibodies, and other biomolecules comprisingthe steps of constructing an appropriately labeled beadset, exposing theheadset to a clinical sample, and analyzing the combined sample/beadsetby flow cytometry. Flow cytometric measurements are used to classify, inreal-time, beads within an exposed beadset and textual explanations,based on the accumulated data obtained during real-time analysis, aregenerated for the user. The '793 patent enables the simultaneous, andautomated, detection and interpretation of multiple biomolecules or DNAsequences in real-time while also reducing the cost of performingdiagnostic and genetic assays. However, the '793 patent does notdescribe how to utilize the technology for diagnosing LSD's.

U.S. patent application Ser. No. 09/956,857 (“the '857 Application”)entitled “Multiple Reporter Read-out for Bioassays” was published onMar. 20, 2003. The '857 Application describes a method for detecting aplurality of reactive sites on an analyte, comprising allowing reactantson an addressable microsphere and the reactive sites to react, formingreactant-reactive site pairs distinguishable by fluorescence intensity.The '857 Application also provides a method for detecting a plurality ofanalytes in a sample using addressable microspheres in combination withone or more reporter reagents. Also provided are a method fordetermining allele zygosity of a genetic locus having two alleles ormore alleles using microparticles, and a method for detecting aplurality of SNPs in nucleic acid molecules. The '857 Application alsoprovides a composition comprising an addressable microsphere carrying atleast two fluorescent reactants capable of forming reactant-analytepairs distinguishable by their fluorescence intensity, and kitscomprising the inventive composition and a plurality of reporterreagents. However, the '857 Application does not describe how to utilizethe technology for diagnosing LSD's. The entirety of each of theapplications or patents listed above is hereby specifically incorporatedby reference.

Accordingly, there is a need for the development of a fast, accurate andeconomical screen for early diagnosis of LSDs, which is amenable toautomation. The ability to identify specific LSD enzymes in an automatedmultiplex assay will have a significant impact on the development of anewborn screening programs, as well as the ability to address a numberof other issues associated with the early diagnosis and treatment ofLSDs. The present invention provides compounds, reagents, and methodsfor a LSD diagnostic multiplex assay.

FIGURES

FIG. 1 shows LAMP-1 levels in plasma from LSD individuals wherein thebox length is the interquartile range that covers 25th to 75thpercentile, the outliers are represented by (circles) each of thesecases represent values between 1.5 and 3 box lengths from the upper orlower edge of the box, and the extreme outlier (stars) are cases withvalues more than 3 box lengths from the upper or lower edge of the box;

FIG. 2 shows saposin C levels in plasma from LSD individuals wherein thebox length is the interquartile range that covers 25th to 75thpercentile, the outliers are represented by (circles) each of thesecases represent values between 1.5 and 3 box lengths from the upper orlower edge of the box; and the extreme outlier (stars) are cases withvalues more than 3 box lengths from the upper or lower edge of the box;

FIG. 3 shows α-Glucosidase in plasma from LSD affected individuals,wherein the box length is the interquartile range that covers 25th to75th percentile, the outliers are represented by (circles) each of thesecases represent values between 1.5 and 3 box lengths from the upper orlower edge of the box, and the extreme outlier (stars) are cases withvalues more than 3 box lengths from the upper or lower edge of the box;

FIG. 4 shows analysis of patient blood spots for LAMP-1 wherein the boxlength is the interquartile range that covers 25th to 75th percentile,the outliers are represented by (circles) each of these cases representvalues between 1.5 and 3 box lengths from the upper or lower edge of thebox, and the extreme outlier (stars) are cases with values more than 3box lengths from the upper or lower edge of the box;

FIG. 5 shows Analysis of patient blood spots for saposin C wherein thebox length is the interquartile range that covers 25th to 75thpercentile, the outliers are represented by (circles) each of thesecases represent values between 1.5 and 3 box lengths from the upper orlower edge of the box, and the extreme outlier (stars) are cases withvalues more than 3 box lengths from the upper or lower edge of the box;

FIG. 6 shows α-Glucosidase protein/activity determination in dried bloodspots, wherein the box length is the interquartile range that covers25th to 75th percentile, the outliers are represented by (circles) eachof these cases represent values between 1.5 and 3 box lengths from theupper or lower edge of the box, and the extreme outlier (stars) arecases with values more than 3 box lengths from the upper or lower edgeof the box;

FIG. 7 shows α-Glucosidase protein distribution in neonates;

FIG. 8 shows the newborn population distribution of LAMP-1 and saposin C

FIG. 9 shows target populations representing each LSD of interestanalyzed;

FIG. 10 shows a microsphere capture sandwich immunoassay having amicrosphere with two spectrally distinct fluorophores, the target LSDcapture antibody and the unique LSD target protein or target antigenbound to the target LSD capture antibody and a reporter molecule;

FIG. 11 shows a list of antibody reagents available for lysosomalproteins for utilization of LSD's screened by multiplex technology;

FIG. 12 shows a calibration curve for α-glucosidase in a microspherebased assay;

FIG. 13 shows multiplexed calibration curves in a microsphere basedassay;

FIG. 14A and FIG. 14B show calibration curves of α-glucosidase usingbead technology and measured using Bio-Plex™ Protein Array system(Bio-Rad);

FIG. 15 shows the multiplex technology having at least a 4-plex forLSD's;

FIG. 16 shows calibration curves for a 4-plex immune quantification oflysosomal proteins;

FIG. 17 shows multiplex analysis of control and MPS I plasma, whereinthe box length is the interquartile range that covers 25th to 75thpercentile, the outliers are represented by (circles) each of thesecases represent values between 1.5 and 3 box lengths from the upper orlower edge of the box, and the extreme outlier (stars) are cases withvalues more than 3 box lengths from the upper or lower edge of the box;

FIG. 18 shows box plots of plasma concentrations of Lamp-1 (A), saposinC (B), α-glucosidase (C) and α-iduronidase (D) from a control group and6 different LSD wherein, the center line within the box represents themedian, the top of the box is the 75^(th) and the bottom of the box isthe 25^(th) percentile, error bars represent the largest and smallestvalues that are not outliers, outliers represented by open circles, arevalues more than 1.5 box lengths from the 75^(th) and 25^(th) percentileand extremes represented by stars are values more than 3 box-lengthsfrom the 75^(th) and 25^(th) percentile.

FIG. 19 shows box plots of concentrations of Lamp-1 (A), saposin C (B),α-glucosidase (C) and α-iduronidase (D) from dried blood spots, thesamples were measured in a control group, a newborn group, and a groupof 3 LSD patients.

FIG. 20 shows target protein markers for LSD screening;

FIG. 21 shows the antibodies and bead regions used for the 7-plex assay;

FIG. 22 shows the calibration curves for each of the protein assays;

FIG. 23 shows the individual and average adult control protein values inthe 7 plex assay obtained for each sample with the standard deviation,minimum and maximum of each group;

FIG. 24 shows the individual and average newborn protein values in the 7plex assay for each sample with the standard deviation, minimum andmaximum of each group;

FIG. 25 shows the Pearson correlation coefficient between each pair ofprotein analytes;

FIG. 26 shows the protein concentrations of the LSD individuals comparedto adult control group;

FIG. 27 shows the protein concentrations of the LSD individuals comparedto the newborn control group;

FIG. 28 shows the multiplex neonatal screening strategy for LSD;

FIG. 29 shows the derivatization of oligosaccharides for MS/MS analysis;

FIG. 30 shows MS/MS analysis of α-mannosidosis urine (Precursor ion scanof m/z 175);

FIG. 31 shows retrospective analysis of HNAcS in newborn blood spots vsblood spot age;

FIG. 32 shows retrospective analysis of HNAc-UA-HNAc-UA in newborn bloodspots;

FIG. 33 shows a summary of retrospective analysis of newborn bloodspots;

FIG. 34 shows protein markers for LSD screening using multiplex assaysfor LSD.

SUMMARY

Lysosomal Storage Disorders (“LSDs”) represent a group of over 40distinct genetic diseases that generally affect young children.Individuals that are affected with a LSD present a wide range ofclinical symptoms that depend upon the specific disorder or a particulargenotype involved. The present invention is generally related to amultiple screening diagnostic for LSD and related diseases. Moreparticularly, this invention pertains to compounds, reagents, andmethods for identifying and quantifying multiple target enzymes andproteins that are used to accurately diagnose a LSD. These targetenzymes and proteins are naturally present in biological fluids ortissues of patients. The invention also pertains to a Multiplexing BeadTechnology for simultaneous screening of specific LSD enzymes.

A first aspect of the current invention is a composition used fordiagnosing a LSD. The composition comprises a capture antibody capableof binding a target antigen, and a microsphere having the captureantibody conjugated to the microsphere. The target antigen is a LSDassociated biomolecule that comprises α-iduronidase, α-glucosidase,saposin C, LAMP-1, LAMP-2, β-glucosidase, α-galactosidase A,iduronate-2-sulphatase, N-acetylgalactosamine 4-sulphatase, galactose6-sulphatase, acid sphingomyelinase, galactocerebrosidase,arylsulphatase A, saposin B, heparan-N-sulphatase,α-N-acetylglucosaminidase, acetylCoA: glucosamine N-acetyltransferase,N-acetylglucosamine 6-sulphatase, β-galactosidase, β-glucuronidase,aspartylglucosaminidase, acid lipase, β-hexosamindase A, β-hexosamindaseB, GM2-acitvator, acid ceramidase, α-L-fucosidase, α-D-mannosidase,β-D-mannosidase, neuraminidase, phosphotransferase, phosphotransferaseg-subunit, palmitoyl protein thioesterase, tripeptidyl peptidase I,cathespsin K, α-galactosidase B, or sialic acid transporter. Themicrosphere having the conjugated capture antibody has a diameter ofabout 5 μm and at least a first fluorophore and a second fluorophore.The first fluorophore being spectrally distinct from the secondfluorophore. The composition may further comprise a detection antibody,wherein the detection antibody is capable of binding the target antigen,but is different from the capture antibody, and the detection antibodyis conjugated to any detectable label known in the art (e.g. afluorescent label).

A second aspect of the current invention comprises a protein profilingmethod for diagnosing a pre-clinical status, or a clinical status of aLSD. The method determines at least a first- and second-target antigenquantity from a target biological sample having an unknown clinicalstatus of LSD. At least a first- and a second-reference antigen quantityare also determined from a reference biological sample having a knownclinical status of LSD. The target antigens are LSD associatedbiomolecules that comprise α-iduronidase, α-glucosidase, saposin C,LAMP-1, LAMP-2, or other biomarkers associated with LSD. By calculatinga target proportion between the first- and second-target antigenquantities, an adjusted target quantity can be assigned. Similarly, anadjusted reference quantity can be assigned by calculating a referenceproportion between the first- and second-reference antigen quantities.The pre-clinical status or the clinical status of an LSD can then bedetermined by comparing a deviation of the adjusted target quantity tothe adjusted reference quantity. In one specific embodiment, the targetbiological sample and the reference biological sample of this method areselected from a cellular extract, blood, plasma, or urine.Alternatively, the second target antigen and the second referenceantigen comprise a biomarker indicator of cell number, organelle number,cell size, organelle size, cell volume, or organelle volume.

A third aspect of the current invention comprises a method fordetermining an amount of at least a first target antigen and at least asecond target antigen indicative of a LSD in a target biological sampleusing a composition of capture antibody microspheres. The methodcomprises incubating at least a first capture antibody microsphere andat least a second capture antibody microsphere with the targetbiological sample forming a capture suspension. The first captureantibody microsphere and the second capture antibody microsphere arethen recovered from the capture suspension. These first- andsecond-recovered microspheres are then hybridized with a first- and asecond-detection antibody, respectively. The first recovered antibodymicrosphere and the second recovered antibody microsphere having a bounddetection antibody can be detected when they are passed through anexamination zone. Data is then collected that relates to one or moremicrosphere classification parameters, the presence or absence of thefirst- or second-detection antibody; and the amount of first- orsecond-detection antibody is quantified. In a specific embodiment, thetarget biological sample is selected from a cellular extract, blood,plasma, or urine. In another specific embodiment, the first targetantigen and second target antigens are each α-iduronidase,α-glucosidase, saposin C or other biomarkers associated with a LSD. Thesecond target antigen may also comprise an indicator of cell number,organelle number, cell size, organelle size, cell volume, or organellevolume.

A fourth aspect of the current invention comprises a method of detectingmultiple LSD target antigens in a sample. The specific subset of LSDantigens comprises α-iduronidase, α-glucosidase, saposin C or otherbiomarkers associated with LSD. The method comprises exposing a pooledpopulation of target capture microspheres to the sample. Each of thetarget capture microspheres have distinct subsets, and each distinctsubset has: (i) one or more characteristic classification parametersthat distinguishes one target capture microsphere of one subset fromthose of another target capture microsphere subset according to apredetermined discriminate microsphere function table, which includesfluorescence emission intensities; and (ii) a distinct capture antibodythat can bind a specific subset of LSD antigens. After the pooledpopulation of target capture microspheres has been exposed to thesample, the exposed pooled population of target capture microspheres ispassed through examination zone. The identity and quantity of eachspecific subset of LSD target antigen of interest is determined, ifpresent, in the sample by (i) collecting data relating to one or moresubsets of target capture microsphere classification parameters thatdistinguishes one target capture antibody microsphere of one subset fromthose of another target capture antibody microsphere subset according toa predetermined discriminate function table, including the fluorescenceemission intensities, (ii) collecting data relating to the presence orabsence of a corresponding subset of specific LSD antigen, (iii)quantifying each corresponding subset of specific LSD antigen on eachsubset of capture antibody microsphere. In a specific embodiment, themethod further comprises adding a pooled population of detectionantibodies to the exposed pooled population of the target capturemicrospheres prior to passing the target capture microspheres throughthe examination zone.

DETAILED DESCRIPTION

Terms:

The term “a” or “an” as used herein in the specification may mean one ormore. As used herein in the claim(s), when used in conjunction with theword “comprising”, the words “a” or “an” may mean one or more than one.As used herein “another” may mean at least a second or more.

The term “animal,” “subject,” or “patient” as used herein may be usedinterchangeably and refers to any species of the animal kingdom. Inpreferred embodiments it refers more specifically to humans.

The term “biomolecule” as used herein is understood to represent thetarget molecule, such as a protein, an antibody, a metabolite, a DNAsequence, an RNA sequence; a biologic with activities used or measuredfor the purposes multiplexing and profiling of target biomolecules, or acombination thereof, for the composition and method of determining LSD,used in administering, monitoring, or modifying an LSD therapy.

The term “clinical status” as used herein refers to patients that arebeing studied or treated by physicians for a LSD.

The term “comprise,” or variations such as “comprises” or “comprising,”as used herein may be used to imply the inclusion of a stated element orinteger or group of elements or integers, but not the exclusion of anyother element or integer or group of elements or integers.

The term “fluorophore” as used herein refers to any fluorescent compoundor protein that can be used to quantify the LSD antigens.

The term “normalize” as used herein refers to bringing a target,reference, or other samples into conformity with a standard, pattern,model, etc. For example, in one embodiment, urine samples from LSDpatients and non-LSD patients were normalized by using a 1 μmolequivalent of creatinine from each sample.

The term “phenotype” as used herein refers to the manifestcharacteristics of an organism collectively, including anatomical andpsychological traits, that result from both its heredity and itsenvironment.

The term “preclinical status” as used herein refers to the period of adisease before any of the clinical symptoms appear.

The term “lysosomal storage disorder (“LSD”) associated biomolecule” asused herein refers to any biomolecule that has been linked to any LSD.In preferred embodiments, a LSD associated biomolecule includes, but isnot limited to: α-iduronidase, α-glucosidase, saposin C, LAMP-1, LAMP-2,β-glucosidase, α-galactosidase A, iduronate-2-sulphatase, α-iduronidase,N-acetylgalactosamine 4-sulphatase, galactose 6-sulphatase, acidsphingomyelinase, galactocerebrosidase, arylsulphatase A, saposin B,heparan-N-sulphatase, α-N-acetylglucosaminidase, acetylCoA: glucosamineN-acetyltransferase, N-acetylglucosamine 6-sulphatase, β-galactosidase,β-glucuronidase, aspartylglucosaminidase, acid lipase, β-hexosamindaseA, β-hexosamindase B, GM2-acitvator, acid ceramidase, α-L-fucosidase,α-D-mannosidase, β-D-mannosidase, neuraminidase, phosphotransferase,phosphotransferase g-subimit, palmitoyl protein thioesterase,tripeptidyl peptidase cathespsin K, α-galactosidase B, or sialic acidtransporter. As shown below, Table 1 indicates some enzyme deficienciesfor LSDs.

TABLE 1 Enzymes deficient in some common lysosomal storage disordersAustralian Disease Clinical Phenotype Enzyme Deficiency PrevalenceGaucher disease types I/II/III Gaucher disease Glucocerebrosidase 1 in57,000 (β-glucosidase) Cystinosis Cystine transporter 1 in 192,000 Fabrydisease Fabry disease α-Galactosidase A 1 in 117,000 Glycogen storagedisease II Pompe disease α-Glucosidase 1 in 146,000Mucopolysaccharidosis type I Hurler/Scheie α-L-Iduronidase 1 in 88,000syndrome Mucopolysaccharidosis type II Hunter syndromeIduronate-2-sulphatase 1 in 136,000 Mucopolysaccharidosis type VIMaroteaux-Lamy N-acetylgalactosamine 4- 1 in 235,000 syndrome sulphataseMucopolysaccharidosis type IVA Morquio syndrome Galactose 6-sulphatase 1in 169,000 Niemann-Pick disease types A/B Niemann-Pick disease Acidsphingomyelinase 1 in 248,000 Globoid cell leucodystrophy Krabbe diseaseGalactocerebrosidase 1 in 201,000 Metachromatic leucodystrophyArylsulphatase A 1 in 92,000 Metachromatic leucodystrophy Saposin BMucopolysaccharidosis type IIIA Sanfilippo syndrome Heparan-N-sulphatase1 in 114,000 Mucopolysaccharidosis type IIIB Sanfilippo syndromeα-N-Acetylglucosaminidase 1 in 211,000 Mucopolysaccharidosis type IIICSanfilippo syndrome AcetylCoA: N- 1 in 1,407,000 acetyltransferaseMucopolysaccharidosis type IIID Sanfilippo syndrome N-Acetylglucosamine6- 1 in 1,056,000 sulphatase Mucopolysaccharidosis type IVB Morquiosyndrome β-Galactosidase Mucopolysaccharidosis type VII Slyβ-Glucuronidase 1 in 2,111,000 Niemann-Pick disease type C1 Niemann-Pickdisease Cholesterol trafficking 1 in 211,000 Niemann-Pick disease typeC2 Niemann-Pick disease Cholesterol trafficking 1 in 2,111,000Aspartylglucosaminuria Aspartylglucosaminidase Cholesterol ester storagedisease Wolman disease Acid lipase 1 in 528,000 GM1-Gangliosidosis typesI/II/III β-Galactosidase 1 in 384,000 GM2-Gangliosidosis type I TaySachs disease β-Hexosaminidase A 1 in 201,000 GM2-Gangliosidosis type IISandhoff disease β-Hexosaminidase A & B 1 in 384,000 GM2-GangliosidosisGM2-activator deficiency Farber Lipogranulomatosis Farber disease Acidceramidase Fucosidosis α-L-Fucosidase >1 in 2,000,000 Galactosialidosistypes I/II Protective protein α-Mannosidosis types I/II α-D-Mannosidase1 in 1,056,000 β-Mannosidosis β-D-Mannosidase Mucolipidosis type ISialidosis types I/II Neuraminidase Mucolipidosis types II/III I-celldisease; Phosphotransferase 1 in 325,000 Mucolipidosis type IIICpseudo-Hurler Phosphotransferase g-subunit polydystrophy Mucolipidosistype IV Unknown Multiple sulphatase deficiency Multiple sulphatases 1 in1,407,000 Neuronal Ceroid Batten disease Palmitoyl protein thioesteraseLipofuscinosis, CLN1 Neuranal Ceroid Batten disease Tripeptidylpeptidase I Lipofuscinosis, CLN2 Neuronal Ceroid Vogt-Spielmeyer Proteinfunction not known Lipofuscinosis, CLN3 disease Neuronal Ceroid Battendisease Protein function not known Lipofuscinosis, CLN5 Neuronal CeroidNorthern Epilepsy Protein function not known Lipofuscinosis, CLN8Pycnodysostosis Cathepsin K Sialic acid storage disease Schindlerdisease α-Galactosidase B Sialic acid storage disease Sialuria; salladisease Sialic acid transporter 1 in 528,000 Prevalence figures quotedfrom Miekle et al., JAMA 281: 249-254 (1999). Prevalence and ratio oflysosomal storage disorders may vary from country to country

The term “reference quantity” as used herein refers to a known,normalized amount of a LSD biomarker in a biological fluid. Thereference quantity is determined from an animal, or group of animalshaving a defined clinical status, preclinical status, or phenotype of aLSD disease. The reference quantity may refer to a table compiled fromvarious animals or groups of animals having correlations betweenrelative amounts of LSD biomarkers in a biological fluid, and a knownclinical status, preclinical status, or phenotype.

Lysosomal Storage Disorders

The LSD's represent a group of over 40 distinct genetic diseases thatgenerally affect young children. Patients are usually born without thevisible features of a LSD, but early stage symptoms can quickly developinto a progressive clinical concern. Although some effective LSDtherapies have been developed, it is paramount that therapy be startedas soon as the LSD has been diagnosed. Unfortunately, a clinicaldiagnosis of a LSD often requires multiple visits to a range ofspecialists requiring time-consuming, invasive, complex, inconvenient;and expensive assays. The current process for an accurate diagnosis ofLSD for a patient not having a family history of LSD can take months toyears, which is unacceptable when effective LSD therapies are neededearlier.

It is generally recognized that the accumulation of storage materials inthe lysosomes of LSD affected individuals will increase fromapproximately 1% to as much as 50% of the total cellular volume. Certainlysosomal proteins are present at altered levels in the LSD affectedindividuals (Meikle et al., 1997; Hua et al., 1998), as indicated inFIGS. 1-6. The values for the individual immunoassays in plasma sampleswere determined as follows and shown in FIGS. 1-6. Unless statedotherwise all regents were of analytical grade and were obtained fromSigma Chemical Company, MO USA. Preparation of recombinant proteins,antibodies and calibration standards for Lamp-1 and saposin C.Recombinant Lamp-1 (minus tail) was isolated from CHO-K1 cells asdetailed in Isaac et al [Isaac E L, Karageorgous L E, Brooks D A,Hopwood J J and Meikle P J. Experimental Cell Research 2000, 254:204-209]. Recombinant Saposin C was a gift from Dr G A Grabowski and wasprepared by the method of Qi and Grabowski [Qi T L and Grabowski G A JBiol Chem 1994, 269: 16746-16753].

The anti Lamp-1 monoclonal antibody (BB6) was generated using intactLamp-1 protein by the method of Carlsson and Fukada [Carlsson S R andFukada M JBC (1989) 264(34): 20526-20531] and 7132 (anti Saposin C)monoclonal antibody was produced using the recombinant protein by themethod described in [Zola H and Brooks D. Techniques for the productionand characterization of monoclonal hybridoma antibodies. In: Hurrell J GR, ed. Monoclonal hybridoma antibodies: techniques and applications.Boca Raton, Fla.: CRC Press, 1982:1-57]. Polyclonal antibodies weregenerated for both Lamp-1 and Saposin C by immunizing separate rabbitswith 200 μg of each recombinant protein per inoculation (fourinoculations in total) based upon the method of Leonova et al, 1996,[JBC 271:17312-20]. All antibodies were purified using 5 ml Hitrap™Protein G affinity column (Pharmacia, Uppsala, Sweden). The polyclonalantibodies were affinity, purified further by column chromatographyusing their respective recombinant proteins coupled to Affi-Gel® 10 Gel(Bio-Rad #153-6046, CA, USA) according to manufacturers instructions.

Blood spot calibrators containing final concentrations of 2000, 1000,500, 250, 62.5 and 0 μg/L for Lamp-1 and saposin C′ were prepared asdetailed in Umapathysivam et al [Umapathysivam K, Whittle A M, RanieriE, Binefloss C, Ravenscroft E M, van Diggelen O P, Hopwood J J andMeikle P J Clin Chem 46(9): 1318-1325 2000]. Two blood spot controlscontaining low (Lamp-1 400 μg/L; saposin 200 μg/L) and high (Lamp-1 800μg/L; saposin C 500 μg/L) protein concentrations were similarlyprepared.

Quantification of Lamp-1 and Saposin C in Dried Blood Spots ContainingEDTA.

Lamp-1 and Saposin C were measured in dried blood spots using one stepthree tier, time-delayed fluorescence immunoassays. Microtiter plates(Labsytems, Helsinki, Finland #95029180) were coated with either BB6 or7B2 at a concentration of 5 μg/L in 0.1 mol/l NaHCO3, pH 8.3 andincubated covered for approximately 16 hrs at 4° C. Plates were washedtwice with wash buffer (0.25 mol/lNaCl, 0.02 mol/l Tris containing0.005% TWEEN® 20 surfactant (BDH, Poole, England) and 0.002%Thiomerosal, pH7.8) Non-specific binding sites on the plates wereblocked by the addition of 100 μl of 0.25M NaCl, 0.02M Tris containing0.5% skim milk powder (Diploma, Bonlac Foods, Victoria, Australia), pH7.8, per well. After a two hour incubation at room temperature, themicrotiter plates were washed twice with 0.25M NaCl, 0.02M Tris pH 7.8and tapped dry before being lyophilized and stored desiccated at 4° C.prior to use.

Standard calibrators, controls and patient dried blood spots were placedin duplicate into the coated microtiter wells with 200 μl of eitherpolyclonal antibody diluted in assay buffer (0.15 mol/l NaCl, 0.05 mol/LTris, 20 μmol/L Diethylene triamine-penta-acetic acid, containing 0.01%TWEEN® 40 surfactant, 0.5% bovine serum albumin (A-9647), 0.05% bovineγ-globulin (G-7516), and 0.05% sodium azide, pH 7.8). The antibodieswere used at a final concentration of 200 μg/L and 400 μg/L for theanti-Lamp-1 and anti saposin C polyclonal respectively. The plates werecovered and incubated at room temperature for one hour with shaking,then placed overnight at 4° C., followed by an hour incubation withshaking at room temperature. The blood spots were removed by suction andthe plates washed six times with wash buffer. After dilution in assaybuffer to final concentration of 0.1 μg/ml, 100 μl of anti rabbiteuropium labeled antibody (Wallac, Finland #AD0105), was added to everywell and incubated for one hour at room temperature with shaking. Afterwashing the plates a final six times with wash buffer, 200 μl of DELFIA®Enhancement solution (Wallac, Finland) was added per well and the platesincubated at room temperature for ten minutes with shaking. Fluorescencewas measured on a DELFIA® 1234 Research Fluorometer (Wallac, Finland).The concentrations of Lamp-1 and Saposin C in the blood spots werecalculated using spline fit curves generated by Multicalc Data Analysissoftware (version 2.4 Wallac, Finland)

FIG. 1 shows the LAMP-1 levels in plasma from LSD individuals that areindicated by the box length being the interquartile range that covers25th to 75th percentile. FIG. 2 shows saposin C levels in plasma fromLSD individuals wherein the box length is the inter-quartile range thatcovers 25th to 75th percentile. FIG. 3 shows α-Glucosidase in plasmafrom LSD affected individuals, wherein the box length is theinter-quartile range that covers 25th to 75th percentile.

Target enzymes can also be detected by individual immunoassays in driedblood spots, as indicated in FIG. 4, FIG. 5, and FIG. 6. For example,FIG. 4 shows analysis of patient blood spots for LAMP-1, wherein the boxlength is the inter-quartile range that covers 25th to 75th percentile.FIG. 5 shows Analysis of patient blood spots for saposin C wherein thebox length is the inter-quartile range that covers 25th to 75thpercentile. FIG. 6 shows α-Glucosidase protein/activity determination indried blood spots, wherein the box length is the inter-quartile rangethat covers 25th to 75th percentile. FIG. 7 shows α-Glucosidase proteindistribution in neonates. FIG. 8 shows the newborn populationdistribution of LAMP-1 and saposin C, and FIG. 9 shows targetpopulations representing each LSD of interest analyzed.

Although certain lysosomal target proteins are present at altered levelsin the affected individuals, the current individual screening assays maybe inaccurate due to variations among individual samples. For example, agiven sample is assumed to contain an average number of lysosomes orwhite blood cells (“WBC”), however variations in these values betweenindividual samples are not typically considered. Thus, variations in anindividual having a deficiency in a particular LSD biomolecule (e.g.lysosomal target protein), but also having an unusually high WBC countor high numbers of lysosomes in the test sample may return an assayresult that is consistent for individuals that do not have a LSD.Consequently, if WBC or high numbers of lysosomes were controlled in thesample preparation a large inaccuracy could be avoided, and a properdiagnosis could be made during the first round of LSD screening.

Determining the quantities of multiple target enzymes increases theaccuracy of diagnosing a specific LSD as compared to any single assay.For example, using immunoquantification assays directed towardidentifying the levels of the lysosome-associated membrane proteins(“LAMPs”), such as LAMP-1 or LAMP-2, in an “at-increased-risk” groupwill identify up to 65% of LSD affected individuals. However, thecombination of LAMP's with one of the saposins increases identificationof LSD affected individuals to approximately 85%. Therefore, a method toidentify two or more biomarkers simultaneously would increase theaccuracy of LSD diagnosis and reduce the time and cost for each assay. AMultiplexing Bead Technology is used to simultaneously detect specificat least 2 LSD target antigens is described below or in Table 1.

Example 1 Multiplexing Bead Technology and Target LSD Proteins

The Multiplexing Bead Technology is built around 3 core technologies.The first is the family of fluorescently dyed microspheres having boundbiomolecules. The second is a flow cytometer with 2 lasers andassociated optics to measure biochemical reactions that occur on thesurface of the microspheres, and the third is a high-speed digitalsignal processor to efficiently manage the fluorescent output. Bio-Rad(Hercules, Calif.), provides a commercially available protein arraysystem called the “Bio-Plex™”. The BioPlex™ protein array systemincludes fluorescently dyed microspheres, a flow cytometer with 2 lasersand associated optics, and a high-speed digital signal processor.However, neither the Bio-Plex™ protein array system nor any othercommercially available systems include any specific biomolecules,methods, compounds, or reagents needed for the simultaneous screening ofspecific LSD enzymes.

The Bio-Plex™ protein array system uses multiplexing technology toenable the simultaneous quantitation of up to 100 different analytes.This technology uses polystyrene microspheres internally dyed withdiffering ratios of 2 spectrally distinct fluorophores. Each fluorophorecan have any of 10 possible levels of fluorescent intensity, therebycreating a family of 100 spectrally distinct bead sets. In a preferredembodiment, the dyed microspheres are conjugated with monoclonalantibodies specific for a target LSD protein or peptide thereof.Although not wanting to be bound by theory, each of the 100 spectrallydistinct bead sets can contain a capture antibody specific for a uniqueLSD target protein. In a multiplexed Bio-Plex™ assay, LSDantibody-conjugated beads are allowed to react with the sample and asecondary LSD antibody, or a detection LSD antibody in a microtiterplate well to form a capture sandwich immunoassay. FIG. 10 shows adrawing of a complete microsphere capture sandwich immunoassay having apolystyrene microsphere (110) with 2 spectrally distinct fluorophores;the target LSD capture antibody (120) bound to the microsphere; a uniqueLSD target protein or target antigen (130) bound to the target LSDcapture antibody; a detection LSD antibody (140); and a detectionmolecule (150). Once the complete microsphere capture sandwichimmunoassay has formed in solution, the immunoassay solution is thendrawn into the Bio-Plex™ array reader, which illuminates and reads thesample. Although not wanting to be bound by theory, there are manyenzyme deficiencies specific for a particular LSD, and some of theseenzymes are shown in Table 1. Specific capture antibodies, and detectionantibodies for the target compounds are available for specific LSD's, asshown in FIG. 11. Additional capture antibodies and detectionsantibodies include: β-glucosidase; α-galactosidase A;iduronate-2-sulphatase; α-iduronidase; N-acetylgalactosamine4-sulphatase; galactose 6-sulphatase; acid sphingomyelinase;galactocerebrosidase; arylsulphatase A; saposin B; heparan-N-sulphatase;α-N-acetylglucosaminidase; acetylCoA: glucosamine N-acetyltransferase;N-acetylglucosamine 6-sulphatase; β-galactosidase; β-glucuronidase;aspartylglucosaminidase; acid lipase; β-hexosamindase A; β-hexosamindaseB; GM2-activator; acid ceramidase; α-L-fucosidase; α-D-mannosidase;β-D-mannosidase; neuraminidase; phosphotransferase; phosphotransferaseg-subunit; palmitoyl protein thioesterase; tripeptidyl peptidase I;cathespsin K; α-galactosidase B; sialic acid transporter.

When a red diode “classification” laser (635 nm) in the Bio-Plex™ arrayreader illuminates a dyed bead, the bead's fluorescent signatureidentifies it as a member of one of the 100 possible bead sets.Bio-Plex™ Manager software correlates each bead set to the assay reagentthat has been coupled to it (for example, a first LSD capture antibodycoupled to bead set #22, and a second LSD capture antibody coupled tobead set #42). In this way the Bio-Plex™ protein array system candistinguish between the different assays combined within a singlemicrotiter well. A green “reporter” laser (532 nm) in the array readersimultaneously excites a third fluorescent dye (phycoerythrin, “PE”)bound to the detection LSD antibody in the assay. Although not wantingto be bound by theory, the amount of green fluorescence is proportionalto the amount of target analyte captured in the immunoassay.Extrapolating the captured amount of target analyte to a standard curveallows quantitation of each LSD analyte in the sample. The digitalsignal processing algorithms provide simultaneous real-time dataacquisition of classification and reporter signal output from thousandsof beads per second, supporting up to 100×96=9,600 analyte measurementsfrom each 96-well plate.

Example 2 Designing and Producing LSD Target Microspheres

The BioPlex Protein Array System was used as one embodiment todemonstrate the type and nature of the reagents necessary for a LSDmultiplex diagnostic assay. Four target proteins (e.g. LAMP-1,α-iduronidase, α-glucosidase, and saposin C) were used to design targetcapture microspheres and target reporter antibodies.

The monoclonal capture antibody for LAMP-1 was BB6 developed andprovided by Sven Carlsson (Carlsson et al., 1989). The monoclonalreporter antibody for α-glucosidase (43D1) was obtained from Pharming,Inc. and has been described (Fransen et al., 1988). The polyclonalreporter antibody for LAMP-1, the rabbit polyclonal reporter antibodyfor saposin C, the sheep polyclonal capture antibody for α-glucosidase,and the monoclonal capture antibody (“7B2”) for saposin C were preparedwithin the Lysosomal Diseases Research Unit at the WCH in Adelaide,Australia using standard techniques, known in the art, and brieflydescribed below. The availability and production of specific monoclonaland polyclonal antibodies are know to one of ordinary skill in the art.Production of the specific antibodies uses in the current examples aregiven below:

Polyclonal Antibodies.

Sheep polyclonal antibody was produced against recombinant proteins. Asheep was injected sub-cutaneously with 2 mg of protein in 1 ml, of anemulsion of phosphate buffered saline (pH 7.4) and complete Freundsadjuvant, followed by four booster injections (2 mg each) withincomplete Freunds adjuvant, each three weeks apart. One week after thelast injection the sheep was bled out and serum collected. Rabbitpolyclonal antibody was produced in the same manner, except 0.2-1.0 mgof protein was used per immunisation. Sheep polyclonal antibody waspurified on a 5 ml Hitrap™ Protein G affinity column (Pharmacia Biotech,Uppsala, Sweden) followed by an affinity column prepared from therecombinant protein used for the immunisation. Recombinant proteinaffinity columns were prepared by coupling 5 mg of the recombinantprotein to 2.5 mL of Affi-gel 10 (Bio-Rad, Hercules, Calif., USA) as permanufacturer's instructions.

Briefly, 5 ml of sheep serum was diluted with 5 ml of phosphate bufferedsaline (pH 7.4) and centrifuged (2200 g, 10 min, 4° C.). The centrifugedserum was passed through a 0.2 μm filter, and then loaded on to theProtein G column at a flow rate of 0.5 mL/min. The column was washedwith phosphate buffered saline, pH 7.4 and the antibody eluted with 0.1mol/L H₃PO₄/NaH₂PO₄, pH 2.5 and immediately neutralised by adding 1.0mol/L Na₂HPO₄ ( 1/10^(th) vol). The protein content was estimated byabsorbance at 280 nm (absorbance=1.4 for 1.0 g/L of protein). The eluatewas diluted four fold and then loaded on to the appropriate recombinantprotein affinity column at the same flow rate. The column was washed andeluted as described for the Protein G column.

Monoclonal Antibodies.

Monoclonal antibodies were produced in Balb/C mice using standardimmunisation protocols (Harlow et al., 1988). Mice were immunised withrecombinant enzyme using established protocols. Plasma cells from theseimmunised mice were fused with P3.653 myeloma cells (Zola et ed., 1982)and the resulting hybridoma cell lines screened for antibodies againstthe recombinant protein by direct FT ISA (Harlow et al., 1988).Monoclonal antibodies were purified from cell culture supernatants byammonium sulfate precipitation followed by affinity purification onHitrap™ Protein G affinity column (Pharmacia Biotech, Uppsala, Sweden).

Coupling Antibodies to Microspheres.

The target capture antibodies were Coupled to Bio-Rad carboxylated(“COOH”) beads as follows: anti-LAW-1 to bead #(17), anti-saposin C tobead #(19), and anti-α-glucosidase to bead #(21). The coupling of thetarget capture antibodies to the polystyrene microspheres was performedusing the BioRad bead coupling kit (Catalog number 171-406001, BioRad,Hercules, Calif.). The Bio-Plex™ amine coupling kit includes 4 ml beadwash buffer, 85 ml bead activation buffer, 135 ml PBS, pH 7.4, 10 mlblocking buffer, 25 ml storage buffer, 105 ml staining buffer, 40coupling reaction tubes. The Bio-Plex™ amine coupling kit provides thebuffers necessary to covalently couple 6-150 kD proteins to 5.5 μm dyedcarboxylated polystyrene beads in under 5 hr. The covalent couple of thetarget capture antibody to the carboxylated polystyrene bead is achievedvia carbodiimide reactions involving the protein primary amino groupsand the carboxyl functional groups bound on the surface of polystyrenebeads. The covalent attachment is permanent, leaving no unbound proteinafter cleanup, even after months of storage. The protein-coupled beadscan then be used in multiplex protein-protein binding studies or in thedevelopment of multiplex assays that can be analyzed with the Bio-Plex™protein array system. The bead yield per coupling reaction isapproximately 80%, or enough protein-coupled beads for two 96-wellmicrotiter plates using 5,000 beads per well.

Once the coupling reaction was completed, the target captureantibody-coupled beads were enumerated and the efficiency of the proteincoupling reaction was validated, according to the manufacturer'sprotocol with modifications. In this procedure, the protein-coupledbeads were reacted with a phycoerythrin (“PE”)-labeled antibody thatbinds to the coupled protein, which was then analyzed using theBio-Plex™ protein array system. This procedure was performed by reactingthe beads with a PE-labeled antibody. Alternatively, a reaction using abiotinylated antibody followed by streptavidin-PE may be used. Althoughnot wanting to be bound by theory, the intensity of the fluorescentsignal of this reaction is directly proportional to the amount ofprotein on the surface of the beads. A successful coupling typicallyyields a mean fluorescent intensity (“MFT”) signal that is greater than2,000. The protein coupling validation procedure provided a rapidrelative assessment of the amount of protein coupled to the beads, butcould not verify the functionality of the protein.

Coupling of the phycoerythrin reporter molecule to the detectionantibodies in the LAMP-1, saposin C and α-glucosidase assays wasachieved using the Molecular Probes (Eugene, Oreg., USA) Protein-ProteinCoupling Kit, as per manufacturer's instructions with modifications.There are several published methods known in the art for preparation ofphycobiliprotein conjugates with antibodies and other proteins.Generally, the coupling chemistry used to crosslink a phycobiliproteinto another protein includes: (a) treating the antibody or other proteinwith a succinimidyl ester maleimide derivative at pH 7.5, which convertssome lysine residues of the antibody to thiol-reactive maleimides; (b)preparing a thiolated phycobiliprotein by reducing the appropriateSPDP-modified phycobiliprotein with dithiothreitol (“DTI”) or withtris-(2-carboxyethyl)phosphine (“TCEP”); (c) mixing the above twodialyzed protein conjugates to yield a stable thioether crosslink; and(d) chromatographically separating the phycobiliprotein conjugates fromthe unreacted proteins.

A calibration curve was generated using liquid calibrator proteins in amicrosphere based assay using calibrator protein capture antibodies andbead sets #17, #19 and #21 respectively (BioRad, Hercules, Calif., USA).FIG. 12 shows a calibration curve for a single assay for α-glucosidase.The detection capability for the amount of calibrator protein present ineach well reaction was linear in the range of 0 to 4 ng/well of theassay. The MFI was the average of the total fluorescence detected forthe beads in the defined bead region. Calibration curves were alsoestablished, using liquid calibrators, for LAMP-1 (open square), saposinC (open circle), and α-glucosidase (open triangle), as shown in FIG. 13.Increased MFI for the α-glucosidase protein, when compared to FIG. 12,is the result of improvements in the capture antibody labeling of themicrospheres and the phycoerythrin reporter labeled antibodies.

FIG. 13 also indicates that the detection capability for a multiplexassay of three calibrators was linear from 0 to 2 ng/well of the assay.The sensitivity of the microsphere assay system was also demonstratedwith the target capture sheep polyclonal antibody for α-glucosidase andbead set (#19) using a biotinylated reporter antibody withstreptavidin-phycoerythrin conjugate (Molecular Probes #S-866). As shownin FIG. 14, α-glucosidase was detectable down to a level of 10 pg/wellusing this assay. FIG. 14A shows the calibration curve in the range0-2.5 ng/well, and FIG. 14B shows the same calibration curve expanded inthe range 0-0.156 ng/well.

Example 3 Four-Plex Assay for the Determination of LAMP-1,α-Iduronidase, α-Glucosidase and Saposin C

A high sensitivity, four-plex assay for target antigens LAMP-1,α-iduronidase, α-glucosidase, and saposin C was developed using themicrosphere technology based upon Luminex LABMAP™ technology. As ageneral illustration, FIG. 15 shows a drawing of a microspherecollection of capture sandwich immunoassays for the 4-plex having: 4spectrally distinct polystyrene microsphere (510-513); 4 target LSDcapture antibody (520-523) bound to the microsphere; 4 unique LSD targetproteins or target antigens and representing saposin, LAMP-1;α-iduronidase and α-gulucosidase (530-533) bound to the correspondingtarget LSD capture antibody; 4 unique detection LSD antibody (540-543);and a detection molecule (550).

Specific Target Capture Microspheres and Target Reporter Antibodies

Specific target capture microspheres and target reporter antibodies wereproduced using antibodies directed against four specific target proteins(e.g. LAMP-1, α-iduronidase, α-glucosidase, and saposin C), as describedabove. The sheep anti-α-iduronidase□ and anti-α-glucosidase polyclonalantibodies were initially purified by ammonium sulphate precipitation.The ammonium sulphate precipitation purified antibodies were furtherpurified using a protein G affinity purification (Amersham Pharmacia 5ml #17-0404-01). The protein G affinity purified antibodies were finallypurified using an Hi trap NHS-activated HP column (Amersham Pharmacia 5ml #17-0717-01) coupled with either a α-iduronidase or α-glucosidaseprotein. The antibodies for anti-LAMP-1, anti-α-iduronidase,anti-α-glucosidase, and anti-saposin C were purified from hybridomasupernatant using protein G affinity purification according tomanufacturer's specifications (Amersham Pharmacia 5 ml #17-0404-01).

Specific target capture microspheres and target reporter antibodies wereproduced using antibodies directed against four specific target proteins(e.g. LAMP-1, α-iduronidase, α-glucosidase, and saposin C). Specifictarget capture microspheres and target reporter antibodies were producedusing antibodies directed against four specific target proteins (e.g.LAMP-1, α-iduronidase, α-glucosidase, and saposin C). The captureantibodies were coupled to microsphere beads by a 2-step carbodiimidereaction according to manufacturers instructions (Bio-Rad, Aminecoupling kit 171-406001). For example, sheep anti-α-iduronidase □and□,anti-α-glucosidase polyclonal antibodies and anti-saposin C monoclonalantibody (7B2) were coupled to dyed polystyrene beads using the antibodyprotein amino group via carbodiimide chemistry according tomanufacturer's instructions at a concentration of 9 μg of IgG to 1.4×10⁶beads.

One with ordinary skill in the art is aware of the several publishedmethods known for efficiently biotinylating antibodies and otherproteins. For example, the purified anti-LAMP-1, anti-α-iduronidase(Id1A), anti-α-glucosidase (43D1), and anti-saposin C (S13C1) monoclonalantibodies were biotinylated using manufacturer's instructions for aFluoReporter® Biotin-XX Protein labeling kit F-2610 purchased fromMolecular Probes (Eugene, Oreg.). Generally, the FluoReporter® Biotin-XXProtein Labeling Kit contains a biotin-XX succinimidyl ester, whichreacts with primary amines of proteins or other biomolecules to formstable biotin conjugates. The long spacer between the biotin and there-active group in biotin-XX succinimidyl ester enhances the ability ofthe conjugated biotin to interact with the relatively deepbiotin-binding sites of avidin and streptavidin. The biotinylatedprotein was purified from the excess biotin using a gel filtrationcolumn. The degree of biotinylation was determined using an avidin-HABAcomplex and a control biotinylated goat IgG.

Development of Four-Plex Assays.

LSD target antigen capture microspheres were diluted in PBS containing1% BSA (assay buffer). The diluted LSD target antigen capturemicrospheres were then added to stock beads in a 96 well filtrationplate (Millipore #MABVS1210), wherein the diluted LSD target antigencapture microspheres and stock beads had a total volume of 1 μl perwell. Each microwell containing the beads was then washed 3 times withPBS containing 0.05% Tween 20 (wash buffer) under vacuum using amanifold (Millipore #MAVM096OR). Standard solutions containing LAMP-1,α-iduronidase, α-glucosidase, and saposin C protein (50 μl) were addedin serial 2-fold dilutions in assay buffer, as indicated. Standards weregenerated by using the recombinant form of each specific target protein.Biotinylated antibodies (50 μl) were added to each well, wherein thefinal concentration of each antibody was 16 ng/well in assay buffer. Theplate was covered and incubated for 2 hours at room temperature withshaking. The wells were washed, incubated with StreptavidinR-phycoerythrin conjugate (Molecular Probes #S-866) (50 ng/well) inassay buffer for 10 minutes at room temperature with shaking. After afinal wash, 125 μl of assay buffer was added per well and the plateshaken for 5 minutes at room temperature. Fluorescence was measuredusing the Bio-Plex™ Protein Array system in combination with theBio-Plex™ software version 2.0 (Bio-Rad, Hercules, Calif.). FIG. 16shows the resulting calibration curves for LAMP-1 (solid square),α-iduronidase (open circle), α-glucosidase (open square), and saposin C(open triangle) of the four-plex assay.

Samples. Plasma and blood samples were collected from infants, childrenand adults. Although plasma samples and dried blood spots were used asexample samples, other suitable sample types are also embodied for thisinvention (e.g. amniotic fluid, cellular extract, urine, etc.) Theplasma and blood spot samples used to demonstrate the four-plex wereobtained from the National Referral Laboratory and Neonatal ScreeningLaboratory Women's and Children's Hospital (Adelaide, Australia) andresearch laboratories at the Lysosomal Disease Research Unit (Adelaide,Australia). Blood collection and blood spotting techniques are wellestablished, and known by one with ordinary skill in the art.

The bead assays were performed in 96 well filtration plates (MilliporeMAV BVS12) and protected from light. Although 96 well filtration plateswere utilized, one with ordinary skill in the art understand that othertypes of sample holders can be used without diverting the scope andspirit of the invention. Plasma samples were diluted in PBS containing1% BSA (Sigma A-9647) pH 7.2 (assay buffer) at a final concentration of3 μl/well. Samples derived from 3 mm dried blood spots were pre-elutedovernight at 4° C. in 100 μl of assay buffer in 96 well low proteinbinding plates (Greiner 655101), wherein 50 μl of each eluted sample wasthen transferred to a filtration plate. Sample assays and standardassays were performed in duplicate with the exception of the newbornsample blood spots, wherein only a single sample for each newborn wasmeasured.

Following sample preparation, the capture antibody beads were preparedfor the multiplex assay. Each individual multiplex assay contained amixture of capture antibody beads for each of the LAMP-1, α-iduronidase,α-glucosidase, and saposin C capture antibody beads describe above.About 5,000 capture antibodies beads were placed in each sample well ofa pre-wetted filtration plate. The mixtures of capture antibody beadswere washed 3 times under vacuum in the filtration plate using a washbuffer (PBS, 0.05% Tween 20, pH 7.2), forming a washed/capture beadmixture. Diluted mixed standards or samples prepared as described abovewere added to the microtiter wells containing the washed/capture beadmixture forming an antigen/bead-set mixture. A mixture of the fourbiotinylated reporter antibodies (i.e. LAMP-1, α-iduronidase,α-glucosidase, and saposin C) was added to the antigen/bead-set mixturecompleting assay components.

The plates were sealed and incubated for about 1 hour at roomtemperature with shaking, then placed at 4° C. overnight under staticconditions. The plates were then incubated at room temperature withshaking for about 1 hour. It will be apparent to one skilled in the artof antibody hybridization that incubation conditions can be modifiedwithout altering the scope and spirit of the invention. Followingincubation, the plates were washed 3 times with wash buffer (PBS, 0.05%Tween 20, pH 7.2) under vacuum. Streptavidin conjugated to phycoerythrin(Molecular Probes S-866) was added to the wells and the plates wereincubated at room temperature for 10 minutes. The plates were placed ina Bio-Plex suspension array system (Bio-Rad) and data was collectedusing Bio-Plex™ Manager software version 3.0 software and counting 100beads/region. Analysis of the data was determined using a Mann-Whitney Utests (MWU) and box plots using the SPSS statistical package Version10.0 (SPSS Inc. Chicago, Ill., USA). Percentile cut offs were generatedusing a standard computer spreadsheet.

Plasma Samples.

The concentrations of LAMP-1, α-iduronidase, α-glucosidase, and saposinC in plasma samples, as determined by the four-plex assay are shown inFIG. 17 and FIG. 18. Briefly, FIG. 17 shows multiplex analysis ofcontrol and MPS I plasma, wherein the box length is the interquartilerange that covers 25th to 75th percentile, the outliers are representedby (circles) each of these cases represent values between 1.5 and 3 boxlengths from the upper or lower edge of the box, and the extreme outlier(stars) are cases with values more than 3 box lengths from the upper orlower edge of the box. FIG. 18 shows box plots of plasma concentrationsof LAMP-1 (A), saposin C (B), α-glucosidase (C) and α-iduronidase (D)from a control group and 6 different LSD. The center line within the boxrepresents the median. The top of the box is the 75^(th) and the bottomof the box is the 25^(th) percentile. Error bars represent the largestand smallest values that are not outliers. Outliers represented by opencircles and are considered values that are more than 1.5 box lengthsfrom the 75^(th) and 25^(th) percentile. The extremes are represented bystars having values more than 3 box-lengths from the 75^(th) and 25_(th)percentile.

Plasma LAMP-1 concentrations (FIG. 18 A) were significantly elevatedabove controls for the LSD samples measured (MWU Test p<0.05). Howeversaposin (FIG. 18 B) was only elevated in the Gaucher plasma (MWU Testp<0.05). Plasma α-iduronidase levels (FIG. 18 D) were significantlydecreased in lysosomal diseases tested with respect to controls (MWUTest p<0.05), except for MPS IIIA. MPS I plasma is normally expected tohave negligible if not zero α-iduronidase levels, however, one of theMPS I plasmas has an exceptionally high level of α-iduronidase, which,although not wanting to be bound by theory, probably result frommistargeting of the protein into circulation. From a screening point ofview this patients plasma would be flagged for further investigation.Pompe plasma was the only disease group with significantly lower (MWUTest p<0.05) α-glucosidase levels (FIG. 18 C) when compared to controlsamples.

Although not wanting to be bound by theory, the pattern of LAMP-1elevation in the 3 disorders as compared to controls observed in theplasma samples was not as apparent in a direct comparison of the targetproteins in the sample blood spots (FIG. 19 A) (e.g. none of thedisorders had elevated LAMP-1). Similarly no disorder was elevated forsaposin C (FIG. 19 B). Although not wanting to be bound by theory, thereis an extremely broad range of these four markers in newborns whencompared to a tighter range of the same 4 markers in blood spots fromolder control individuals (i.e. age range 6 months to 47 years). Thebroad range of absolute levels of the marker protein in infants hindersa defined standard of “elevated” levels of Lamp-1 and saposin C innewborns. Additionally, there was no detectable α-iduronidase andnegligible α-glucosidase protein for MPS I and Pompe diseaserespectively (FIG. 19 C and FIG. 19 D) as compared to the control groupin blood spots (MWU Test p<0.001). Some of the newborn controlα-iduronidase levels appear to overlap with the zero levels found in MPSI samples but the lowest level for α-iduronidase in newborns was 0.243ug/L. In contrast to absolute marker measurements, the multiplex allowseach protein to be compared using ratios. For example, there was onefour month old Pompe patient who had α-glucosidase blood spots levels inthe lower range of the control group (FIG. 19 C), this patient wouldhave been missed in a typical screening program if the determinedcut-offs used only absolute protein levels. However, using the ratioswith the multiplex data, α-glucosidase can be compared against eithersaposin C (e.g. ratio of 0.271) or LAMP-1 (e.g. ratio of 0.019), wherebyflagging this patient as an affected in the 2^(nd) percentile. Similarratio values for the older control range were about 0.339 and about0.021 for α-glucosidase/saposin C and α-glucosidase/LAMP-1 respectively.

The multiplex data generated for Pompe patients was used to produce aratio of α-glucosidase to Lamp-1, and this ratio could distinguish 3/3Pompe plasma samples and 9/9 Pompe blood spot samples from thecorresponding plasma and blood spot samples from non-LSD patients.Similarly, the MPS I multiplex ratio data for α-iduronidase to LAMP-1was below the 2^(nd) percentile cut-off for 16/17 plasmas and 4/4 bloodspots. As mentioned previously the one rogue MPS I plasma that does notfit the pattern, but still have been flagged as suspicious due to thevery high α-iduronidase levels.

Although not wanting to be bound by theory, the specific example of a4-plex assay supports the invention that a multi-plex assay combinedwith protein profiling of two or more lysosomal proteins improves thedetection of MPS I and Pompe affected individuals in both plasma andblood spots. Determination of protein profiles that look at two, three,four or more than four-protein concentrations or corresponding ratiosgive even more discriminating power to the LSD multiplex assay. Oneaspect of this invention allows the ratios of LAMP-1 and saposin C to beused as markers to normalize the population for the lysosomal content ofthe patient sample. For such disorders, these proteins profiles provideadditional discriminatory power by showing an increase in concentrationrelative to the non-disease state. Multiplex technology improves thedetection rate for most LSD and has an application in newborn screeningprograms for these diseases.

As shown in the above examples of the multiplex concept combined withthe protein profile/fingerprint concept, there are many ways the profilecan be analyzed. Levels of proteins, ratios of proteins and discriminateanalysis have been described, but other examples could include the useof neural networks. Therefore, it will be readily apparent to oneskilled in the art that various substitutions and modifications may bemade in the invention disclosed herein without departing from the scopeand spirit of the invention.

Example 4 7-Plex Lysosomal Protein Profile Assay

Protein markers for several LSD disorders are shown in FIG. 20. A 7-plexassay for target antigens LAMP-1, saposin C, α-iduronidase,α-glucosidase, α-galactosidase, β-glucosidase andN-acetylgalactosamine-4-sulphatase was developed using the microspheretechnology based upon Luminex LABMAPT™ technology.

Specific Target Capture Microspheres and Target Reporter Antibodies.Specific target capture microspheres and target reporter antibodies wereproduced using antibodies directed against the seven specific targetproteins (e.g. LAMP-1, saposin C, α-iduronidase, α-glucosidase,α-galactosidase, β-glucosidase and N-acetylgalactosamine-4-sulphatase),and the coupling method as outlined above in Example 3. Briefly, thecapture antibodies for LAMP-1, saposin C, α-iduronidase, α-glucosidase,α-galactosidase, β-glucosidase and N-acetylgalactosamine-4-sulphatasewere coupled to microsphere beads by a 2-step carbodiimide reactionaccording to manufacturers instructions (Bio-Rad, Amine coupling kit171-406001). Reporter antibodies were biotinylated according tomanufacturers instructions (Molecular Probes, FluoroReporter Biotin-XXprotein labelling kit F-2610). The recombinant form of each protein weregenerated and used as standards. The dried blood spots that werecollected from newborns, children and adults and used in this study weresamples submitted to the National Referral Laboratory and NeonatalScreening Laboratory Women's and Children's Hospital. Additional sampleswere collected from within the Lysosomal Disease Research Unit. FIG. 21shows the antibodies and bead regions used for the 7-plex assay.

Sample Preparation and Method for Multiplexed Assays (7-Plex).

A 3 mm dried blood spots were pre-eluted in 130 μl of filtered (0.2 μm)PBS containing 0.5% BSA (Sigma A-9647), 0.05% γ-globulin (Sigma G-7516)and 0.05% Tween 20, pH7.2, (assay buffer) for 1 hour at room temperaturewith shaking, followed by 16 h at 4° C. in 96 well, low protein bindingplates (Greiner 655101). The blood spots were incubated a further 1 hourat room temperature with shaking and 100 μl of each eluted sample wasused for the multiplex assay. Bead assays were performed in 96 wellfiltration plates (Millipore MAB VNS1250) sealed and protected fromlight. Samples and standards were performed in duplicate except for thenewborn blood spots where only single samples were used.

Antibody coated beads (5,000/well) for each individual assay were mixedand placed into pre-wetted filtration plates and the supernatant removedby vacuum. Diluted pre-mixed standards or samples were added to thebeads followed by the 7 pre-mixed biotinylated reporter antibodies. Theplates were incubated for 1 hour at room temperature with clinking, thenplaced at 4° C. overnight. After a further 1 hour incubation at roomtemperature with shaking, the plates were washed 3 times with filtered(0.2 μm) PBS containing 0.05% Tween 20, pH 7.2 (wash buffer) undervacuum. Streptavidin conjugated to phycoerythrin (Molecular ProbesS-866) was diluted in assay buffer (1.5 ug/mL) and added to the wells(100 μl/well), then the plates were incubated at room temperature withshaking for 10 minutes. The plates were then read on the Bio-Plexsuspension array system (Bio-Rad) using version 3.0 software andcounting 100 beads/region.

Results for Multiplexed Assays (7-Plex).

Control blood spots from 12 adult and 28 newborn control individualswere assayed for the 7 lysosomal proteins; LAMP-1, saposin C,α-iduronidase, α-glucosidase, α-galactosidase, β-glucosidase andN-acetylgalactosamine-4-sulphatase. FIG. 22 shows the calibration curvesfor each of the protein assays. FIG. 23 shows the individual and averageadult control protein values in the 7 plex assay obtained for eachsample with the standard deviation, minimum and maximum of each group.FIG. 24 shows the individual and average newborn protein values in the 7plex assay for each sample with the standard deviation, minimum andmaximum of each group. Standard deviation, minimum and maximum are alsopresented as multiples of the mean (MOM). Comparison of the standarddeviation, minimum and maximum MOM values for the adult and newborngroups show that the newborn group has a wider range than the adultgroup.

FIG. 25 shows the Pearson correlation coefficient between each pair oftarget protein analytes. With the exception of α-iduronidase, the targetantigens showed a significant correlation to the other target antigens.

Dried blood spot samples from 16 LSD affected individuals representing 5different disorders were also analysed with the 7-plex protein profile.The results of these analyses are shown in FIG. 26 (compared to theadult control group) and FIG. 27 (compared to the newborn controlgroup). The LSD patients were clearly distinguished from the controlgroups.

Example 5 Multiplex Method to Screen the Newborn Population for MajorLSD's

A general multiplex neonatal screening strategy for LSD is illustratedin FIG. 28. A neonatal screening strategy for LSD's can be customizeddepending upon the geographic region and LSD prevalence. For example,the following 14-Plex is an example of an assay suitable for use inNorth America and Europe. Twelve specific LSDs were chosen because oftheir relatively high prevalence in North America and Europe, togetherwith the availability of effective therapies that would benefit fromearly diagnosis. A multiplex assay for the following 14 target proteinscan test for the associated LSD that is shown in parentheses: LAMP-1(generic LSD), saposin C (generic LSD), α-glucosidase (Pompe),α-galactosidase A (Fabry), glucocerebrosidase or β-glucosidase(Gaucher), α-iduronidase (MPS I), iduronate-2-sulphatase (MPS II),heparan-N-sulphatase (MPS IIIA), α-N-acetylglucosaminidsse (MPS MB),galactose-6-sulphatase (MPS IVA), β-galactosidase orgalactocerebrosidsse (Krabbe), galactose-3-sulphatase (MLD),sphingomyelinase (Niemann-Pick A/B) andN-acetylgalactosamine-4-sulphatase (MPS VI).

The protein profiling multiplex technology enables combinations of LSDtarget antigens to be modified as LSD treatment methods improve, as newLSD are identified, or screening needs change in different geographicareas. Antibodies to each of the 14 LSD target antigens are needed forthis 14-plex assay.

The present invention improves the accuracy and detection of each of theLSD's in a single multiplex assay. The target antigens LAMP-1 andsaposin C are used as markers to normalize the population for thelysosomal content of the patient sample. For some disorders, theseproteins may provide additional discriminatory power by showing anincrease in concentration relative to the non-disease state. Bycalculating the ratio of these proteins to the individual proteinsdeficient in each LSD, greater discriminatory power can be attained.This concept can be extended beyond the calculation of ratios ofindividual proteins to the determination of protein profiles thatencompasses many different target antigen protein concentrations for agiven sample. The use of discriminate analysis or other statisticalmethods can provide improved discrimination between control and affectedpopulations.

Protein profiling will improve the sensitivity and specificity ofdetermining an LSD, wherein false negatives can be optimized to asensitivity of 0-20% for most LSD's and false positives can be predictedbetween 0.1% and 0.01%. Additionally, confirmation assays can beperformed on all positive assays prior to recalling the patient.Confirmation testing for LSD type following Multiplex protein profilingcan be completed by methods such as specific enzyme analysis, substratestorage analysis, or genotyping. Enzyme analysis comprises immunecapture assay for specific lysosomal enzymes that are performed on asecond blood spot. The substrate storage analysis comprisesoligosaccharide and glycolipid analysis performed on second and thirdblood spots. Genotyping from dried blood spots comprises screening forcommon mutations where appropriate on a further blood spots.

It is understood that proteins characteristic of other LSD types can bereplaced, or added to the 14 target antigen lysosomal proteins listedabove and that such modifications may depend on the frequency ofindividual LSD's for particular geographic regions. For example, therelative prevalence of individual LSD's is different in North America,Japan and China. It is also understood that other biomolecules canrepresent the specific LSD target antigens or target molecules, such asantibodies, DNA sequences or RNA sequences or protein activities may beused or measured for the purposes multiplexing and profiling of targetbiomolecules this invention.

Example 6 Developing a Multiplex Profiles for a LSD

In one embodiment of the invention a series of at least two lysosomalproteins (e.g. α-glucosidase, β-glucosidase, α-galactosidase,α-iduronidase, iduronate-2-sulphatase andN-acetylgalactosamine-4-sulphatase, etc.) are multiplexed. Samples froma control population (n≧100) are analyzed with the multiplexed assay todetermine the normal range for each of the analytes. Each analyte isnormalized to general lysosomal markers (e.g. LAMP-1 and saposin C) inaddition to the other specific markers to produce a series of ratios, ora fingerprint table. These ratios are then used to provide a profile ofthe control population. Samples from, a target population (Pompe,Gaucher, and other LSD affected individuals) (n≧20) are analyzed and theresults normalized as described in previous examples. The specificratios that best differentiate the control and target populations arethen utilized develop a specific profilelfingerprint of the LSD diseasestate.

Example 7 Multiplex Profiles for Specific a LSD

In one embodiment of the invention a series of at least two lysosomalproteins (e.g. α-glucosidase, β-glucosidase, α-galactosidase,α-iduronidase, iduronate-2-sulphatase andN-acetylgalactosamine-4-sulphatase, etc.) are multiplexed and utilizedas a specific disease diagnostic (e.g. Pompe, Gaudier, Fabry, MPS,Niemann-Pick, Krabbe, etc.). Samples from a control population ofpatients are analyzed with the specific LSD multiplexed assay todetermine the normal range for each of the analytes in the controlpopulation. Each analyte is normalized to the general lysosomal markers(e.g. LAMP-1 and saposin C) in addition to the other specific markers toproduce a series of ratios. These ratios are then used to provide aprofile of the control population. Samples from a target population ofpatients (e.g. Pompe, Gaudier, Fabry, MPS, Niemann-Pick, Krabbe, etc.)are also analyzed to determine the diseased state reference range ofeach analyte in a target disease population. The level of each analytein the target population is identified as being elevated, decreased orunchanged, relative to the control population. This provides a proteinprofile or fingerprint for the target disease state. Target populationsrepresenting each LSD of interest can be analyzed by this method andspecific profiles/fingerprints can be obtained. Samples from patientswith an unknown TSD clinical status are then analyzed and the resultingpatterns compared with the available target protein profiles to identifythe specific LSD disease.

Example 8

Multiplex Profiles for LSD Disease Progression and Therapy Monitoring

At least two lysosomal proteins (e.g. LAMP-1, saposin C, α-glucosidase,β-glucosidase, α-galactosidase, α-iduronidase, iduronate-2-sulphataseand N-acetylgalactosamine-4-sulphatase, etc) are multiplexed. Samplesfrom a control population are analyzed with the multiplexed assay todetermine the normal range for each of the analytes. Samples from apopulation of individuals affected with a specific LSD (e.g. Pompe,Gaucher, Fabry, MPS, Niemann-Pick, Krabbe, etc.) in are also analyzed todetermine the reference range of each analyte in the LSD affectedpopulation. The two sets of data are used as a training data set toperform discriminate analysis. This discriminate analysis will allow theidentification of the LSD disease affected individuals from the controlpopulation and classification for each LSD patient that is correlated tothe disease severity (phenotype), or provide a prediction of phenotype(disease progression) in asymptomatic patients. Samples taken from a LSDaffected individual at different times during the course of therapy areanalysed. The discriminate function is used to determine the degree ofnormalisation of the protein profile for that individual (i.e how closedoes it approach the control profile) and thereby monitor the efficacyof therapy.

Example 9 Multiplex Profiles for Pompe

In one embodiment of the invention a series of at least two lysosomalproteins (e.g. α-glucosidase, β-glucosidase, α-galactosidase,α-iduronidase, iduronate-2-sulphatase andN-acetylgalactosamine-4-sulphatase, etc.) are multiplexed and utilizedas a specific disease diagnostic for Pompe disease. Samples from acontrol population (n≧100) of patients are analyzed with the specificLSD multiplexed assay to determine the normal range for each of theanalytes in the control population. Each analyte is normalized to thegeneral lysosomal markers (e.g. LAMP-1 and saposin C) in addition to theother specific markers to produce a series of ratios. These ratios arethen used to provide a profile of the control population. Samples from atarget population of patients Pompe (n≧20) are also analyzed todetermine the LSD state reference range of each analyte in Pompe diseasepopulation. The level of each analyte in the Pompe population isidentified as being elevated, decreased or unchanged, relative to thecontrol population. This provides a protein profile or fingerprint forthe Pompe disease state. Target populations representing each level ofPompe severity of interest can be analyzed by this method and specificprofiles/fingerprints can be obtained. Samples from patients with anunknown LSD clinical status are then analyzed and the resulting patternscompared with the available target protein profiles to identify thespecific Pompe LSD disease. Additionally, the discriminate function canbe used determine the degree of normalization of the protein profile forthat individual (i.e. how close do the values approach the controlprofile) and thereby monitor the efficacy of a therapy.

Example 10 Multiplex Newborn Screening

In one embodiment of the invention a series of lysosomal proteins(LAMP-1, saposin C, α-glucosidase, β-glucosidase, α-galactosidase,α-iduronidase, iduronate-2-sulphatase andN-acetylgalactosamine-4-sulphatase) are multiplexed. Samples (e.g.,dried blood spots) from newborns in a given population are analyzed fora specific LSD (e.g. none, Pompe, Gaucher, Fabry, MPS, Niemann-Pick,Krabbe, etc.) based on protein profiles/fingerprints of discriminatefunctions as described in Examples 5-9 above. The newborns are thenassigned a probability of being affected by a LSD, wherein furthertesting may be required for newborns verification.

Example 11 Multiplex and Cancer

At least two lysosomal proteins (e.g. LAMP-1, saposin C, α-glucosidase,β-glucosidase, α-galactosidase, α-iduronidase, iduronate-2-sulphataseand N-acetylgalactosamine-4-sulphatase, etc) or cancer antigens aremultiplexed. Samples from a control population are analyzed with themultiplexed assay to determine the normal range for each of theanalytes. Samples from a target population (e.g. cancer affectedindividuals) are also analyzed to determine the reference range of eachanalyte in this population. The two sets of data are used as a trainingset to perform discriminate analysis and identify a discriminatefunction that will enable the separation of the cancer affectedindividuals from the control population. The discriminate function isthen used to identify patients having an unknown protein profileconsistent with the particular cancer under investigation. Thisembodiment thereby provides early identification of the cancer.

Multiplex LSD protein profiling provides solutions to many issuesrelating to newborn screening assays. For example, multiplex LSD proteinprofiling provides sensitivity and specificity required to diagnose aspecific selection of LSD disorders to be screened, wherein additionallysosomal proteins can be added if needed. Multiplex LSD proteinprofiling also provides a platform technology to undertake screening forother LSD populations (e.g. Renal/cardiac clinics for Fabry disease;accociation of Fabry disease w/end-stage renal failure, and associationof Fabry disease w/idiopathic cardiomyopathy, muscle fatigue/sorenessfor adult Pompe disease, and altered lysosomal function and proteinlevels in some types of cancers). Multiplex LSD protein profiling isalso flexable to incorporate non-lysosomal protein markers (e.g. thyroidstimulating hormone, immunoreactive trypsin and others.)

This invention comprises lysosomal protein profiling for LSD, whichencompasses the use of protein marker ratios using existing LSD targetmarkers that are increased with LSD, and the use additional LSD targetmarkers that are decreased with LSD. Protein profiling also utilizes aratio of the LSD target markers to improve discrimination. Some LSDmarkers can be used to correct for lysosome/leukocyte levels.Additionally ratio specific markers (e.g. LAMP-1) can be utilized tocorrect for different lysosomal content and other ratio markers can beutilized to correct for white cell content (e.g. CD45). Protein profilesincorporate different proteins markers that are measured to improvediscrimination. Although Multiplex bead technology has been used as aspecific example, other methods of multiple LSD target proteinmeasurements can be utilized to perform protein profiling. Such methodsdo not deviate from the spirit and scope of the claimed invention.

REFERENCES CITED

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

U.S. Patent Documents

-   U.S. Pat. No. 6,449,562 (“the '562 patent”) entitled “Multiplexed    Analysis of Clinical Specimens Apparatus and Method,” having    Chandler et al. listed as inventors was issued on Sep. 10, 2002.-   U.S. Pat. No. 6,524,793 (“the '793 patent”) entitled “Multiplexed    Analysis of Clinical Specimens Apparatus and Method,” having    Chandler et al. listed as inventors, was issued on Feb. 25, 2003.-   U.S. patent application Ser. No. 09/956,857 (“the '857 Application”)    entitled “Multiple Reporter Read-out for Bioassays” was published on    Mar. 20, 2003.-   PCT Application AU03/00731 entitled “identification of    Oligosaccharides and their Use in the Diagnosis and Evaluation of    Mucopolysaccharidoses and Other Related Disorders,” having Hopwood    et al., listed as inventors, filed on Jun. 13, 2003

Other Publications

-   Carlsson, S. R., M. Fukuda, Structure of human lysosomal membrane    glycoprotein 1, Assignment of disulfide bonds and visualization of    its domain arrangement, J. Biol. Chem. 264:20526-20531 (1989).-   Fransen, J. A., L. A. Ginsel, P. H. Cambier, J. Klumperman, R. P.    Oude Elferink, J. M. Tager, immunocytochemical demonstration of the    lysosomal enzyme alpha-glucosidase in the brush border of human    intestinal epithelial cells, Eur J Cell Biol 47:72-80 (1988).-   Harlow, E., D. Lane, Antibodies, A laboratory manual, Cold Spring    Harbor Laboratory (1988).-   Hua, C. T. et al., Evaluation of the lysosome-associated membrane    protein LAMP-2 as a marker for lysosomal storage disorders, Clin.    Chem. 44(10): 2094-2102 (1988).-   Isbrandt, D., G. Arlt, D. A. Brooks, J. J. Hopwood, K. von Figura,    and C. Peters, Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome):    Six unique arylsulfatase B gene alleles causing variable disease    phenotypes, Am J Hum Genet. 54(3): 454-63 (1994).-   Meikle et al., Prevalence of lysosomal storage disorders, JAMA 281:    249-254 (1999).-   Neufeld, E. F. and J. Muenzer, The mucopolysaccharidoses, The    Metabolic & Molecular Basis of Inherited Disease, 7^(th) Edition.,    pp. 2465-2494 (1995).-   Umapathysivam, K., J. J. Hopwood, P. J. Meikle, Determination of    acid alpha-glucosidase activity in blood spots as a diagnosis for    Pompe Disease, Clin. Chem. 47(8): 1378-1383 (2001).-   Zola, H., D. Brooks, Techniques for the Production and    Characterization of Monoclonal Hybridoma Antibodies, Monoclonal    Hybridoma Antibodies: Techniques and Applications (1982).

The claims defining the invention are as follows:
 1. A composition used for diagnosing a lysosomal storage disorder (“LSD”) comprising: a set of capture antibodies conjugated to microspheres, each microsphere having a different specific capture antibody and having at least a first fluorophore and a second fluorophore wherein the first fluorophore and the second fluorophore are spectrally distinct, wherein the set of capture antibodies comprises antibodies capable of binding to α-iduronidase, α-glucosidase, LAMP-1, and saposin C; and wherein each microsphere conjugated to a specific capture antibody has a specific ratio of the first fluorophore to the second fluorophore; and wherein each microsphere conjugated to a specific capture antibody is spectrally distinct from a microsphere conjugated to a different capture antibody.
 2. The composition of claim 1, further comprising a detection antibody, wherein the detection antibody is capable of binding the target antigen, but is different from the capture antibody; and the detection antibody is conjugated to a detection label.
 3. The composition of claim 1, wherein the microsphere has a diameter of about 5 μm.
 4. The composition of claim 1, wherein the LSD is selected from the group consisting of Fabry; Mucopolysaccharidosis type I (“MPS I”); Mucopolysaccharidosis type II (“MPS-II”); Mucopolysaccharidosis type III (“MPS-III’); Mucopolysaccharidosis type IV (“MPS-IV”); and Glycogen storage disease II (“Pompe”).
 5. The composition of claim 1, wherein the LSD is selected from the group consisting of Gaucher disease types I/II/III; Cystinosis; Mucopolysaccharidosis type VI; Mucopolysaccharidosis type IVA; Niemann-Pick disease types A/B; Metachromatic leucodystrophy; Metachromatic leucodystrophy; Mucopolysaccharidosis type IIIA; Mucopolysaccharidosis type IIIB; Mucopolysaccharidosis type IIIC; Mucopolysaccharidosis type IIID; Mucopolysaccharidosis type VII; Mucopolysaccharidosis type IVB; Niemann-Pick disease type C1; Niemann-Pick disease type C2; Cholesterol ester storage disease; Aspartylglucosaminuria; GM1-Gangliosidosis types I/II/III; GM2-Gangliosidosis type I; GM2-Gangliosidosis type II; GM2-Gangliosidosis; Farber Lipogranulomatosis; β-Mannosidosis; Galactosialidosis types I/II; α-Mannosidosis types I/II; β-Mannosidosis; Mucolipidosis type I; Mucolipidosis types II/III; Mucolipidosis type IIIC; Mucolipidosis type IV; Multiple sulphatase deficiency; Neuronal Ceroid Lipofuscinosis, CLN1; Neuronal Ceroid Lipofuscinosis, CLN2; Neuronal Ceroid Lipofuscinosis, CLN3; Neuronal Ceroid Lipofuscinosis, CLN5; Neuronal Ceroid Lipofuscinosis, CLN8; Pycnodysostosis; and Sialic acid storage disease. 